1. Field of the Invention
The present invention in the field of biochemistry, molecular genetics and medicine related to two newly discovered mutant amyloid precursor proteins (APP) and amyloid peptides that are associated, respectively, with Familial British Dementia and Familial Danish Dementia. Transgenic animals expressing these mutant proteins have progressive neurologic disease characterized by both behavioral and neuropathological changes as compared to nontransgenic age-matched controls. These animals are used to screen agents as potential therapeutics for any of a number of progressive degenerative neurologic syndromes characterized by neurofibrillary tangles and dementia. The invention is exemplified by transgenic mice which overexpress two types of mutant precursor protein (ABriPP and ADanPP) in brain tissue under control of tissue-specific gene regulatory sequences.
2. Description of the Background Art
Familial British Dementia (FBD)
FBD, previously designated familial cerebral amyloid angiopathy-British type (Plant, G T et al., Brain 113:721-747 (1990)), is an autosomal dominant condition characterized by dementia, progressive spastic tetraparesis and cerebellar ataxia with onset in the fifth decade of life (Altschul, S F et al., Nucleic Acids Res. 25:3389-3402 (1997); Worster-Drought, C et al., J. Neurol. Psychopathol. 14:27-34 (1933); Worster-Drought, C et al., Brain 63:237-254 (1940); Worster-Drought, C et al., Brain 67:38-43 (1944)). Pathological features include severe widespread cerebrovascular amyloidosis in the brain and spinal cord, nonneuritic amyloid plaques affecting cerebellum, hippocampus, amygdala and occasionally cerebral cortex, periventricular white matter changes, perivascular amyloid plaques and neurofibrillary degeneration in hippocampal neurons. The autosomal dominant mode of inheritance have been confirmed by the uninterrupted transmission from one generation to the next and by the segregation and sex ratios in a large family with more than 200 members encompassing seven generatons (Plant, G T et al., Brain 113:721-747 (1990)).
The biochemical basis of the disorder has remained elusive. It has been reported as an atypical form of familial Alzheimer""s disease (Corsellis, J et al., Brain 77:571-587 (1954); Aikawa, H et al., Ann. Neurol. 17:297-300 (1985)), as an example of Gerstmann-Strxc3xa4ussler syndrome (Masters, C et al., Brain 104:535-558 (1981); Keohane, C et al., J. Neurol. Neurosurg. Psych. 48:1175-1178 (1985); Courten-Myers, G et al. Neurology 37:269-275 (1987); Pearlman, R L et al., Neurology 38:1249-1254 (1988)) and also regarded as a specific form of primary congophilic angiopathy (Vinters, H, Stroke 18:311-324 (1987)).
Classification attempts based on immunohistochemical analysis failed to demonstrate specific staining of the amyloid deposits with a large set of antibodies directed toward known amyloid molecules, although the lesions were immunoreactive for several amyloid associated proteins, i.e. apolipoproteins E and J and serum amyloid P-component (Ghiso, J et al., J. Neurol. Sci. 129:74-75 (1995)). Recently, C-terminal fragments of xcex1- and xcex2-tubulin were reported to be associated with the amyloid lesions although the identification of the major component of the amyloid deposits remained unsettled (Baumann, M H et al., Biochem. Biophys. Res. Commun. 219:238-242 (1996)).
Familial Danish Dementia (FDD)
FDD is a familial condition characterized by progressive development of cataract and other ocular symptoms, hearing impairment, varying neurological symptoms, and dementia, usually complicated by paranoid reactions and occasional disturbances of consciousness.
The first family described with this disease originated from the Djursland peninsula northeast of the city of Aarhus, Denmark (Strxc3x6mgrem E., xe2x80x9cHeredopathia ophthalmo-oto-encephalica.xe2x80x9d In: Vinken P. J. et al., eds. Handbook of Clinical Neurology. Vol 42. Amsterdam: Elsevier 1981, pp 150-152). Cataract formation seems to be the first symptom, appearing before the age of 30. Impaired hearing tends to appear 10-20 years after the ocular symptoms. Ocular hemorrhages were also observed in some cases. Ataxia of a cerebellar type starts shortly after age of 40. These patients have intention tremor of the trunk and all four limbs as well as hypotonia with pendular reflexes. Paranoid psychosis usually develops after the age of 50, followed by dementia. Most affected individuals die in their 50""s and 60""s.
At autopsy, a uniform, diffuse atrophy of all parts of the brain is clearly observed in all patients. Histological examination shows a very severe chronic, diffuse encephalopathy, expressed most intensely in the cerebellum, the cerebral cortex and the white matter. Cranial nerves are extremely thin and nearly completely demyelinated. Widespread amyloid angiopathy is seen in blood vessels of the cerebrum, including the choroid plexus, cerebellum, retina and spinal cord. Neuritic plaques and sparse neurofibrillary tangles are present in the hippocampus which also shows some neuronal loss. The cerebral white matter also contains some ischemic lesions. As in FBD, described above, dementia is also attributed to both, vascular and parenchymal degenerative lesions. However, the nature of the amyloid deposits and the genetic defect remains unknown.
A number of investigators have produced transgenic mice that are intended to serve as model for Alzheimer""s disease (AD), for example U.S. Pat. No. 5,387,742, U.S. Pat. No. 5,612,486, U.S. Pat. No. 5,811,633, U.S. Pat. No. 5,849,999 and U.S. Pat. No. 5,877,399. Even as AD models, each of these has shortcomings. None of these transgenic mice models express the gene associated with FBD or FDD. Moreover, none of them provide the combination of neuropathological changes desired in a robust model of human degenerative dementias, namely amyloid deposition and neurofibrillary tangles and neuronal degeneration.
Key embodiments of the present invention are designed to overcome this deficiency, provide a useful animal model not only of the two specific familial dementias described, but more importantly, to serve as general model for a group of human degenerative neurological diseases that include dementia.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
The present invention is directed to an isolated wild-type amyloid precursor protein, BriPP, or a mutant ABriPP or ADanPP, the sequences and structures of which are described herein. Also included is a peptide that is a fragment of the protein, more preferably the mutant fragment that is the amyloid peptide, a 34 mer from the C-terminus of the mutant protein. In addition to the proteins and peptides, the invention includes functional derivatives such as amino acid sequence variants or chemical derivatives of the wild-type or mutant protein or peptide.
The present invention is directed to an antibody, polyclonal or monoclonal (mAb), specific for the mutant peptide described above, and to immunoassays to detect the mutant protein or peptide, or to detect the presence of the antibody in a sample.
In another embodiment, the invention provides DNA molecules encoding the wild-type or mutant protein or peptide. Preferably, the DNA is an expression vector wherein the coding sequence is operatively linked to a promoter and control sequences that result in selective expression of the DNA in the brain when the DNA is provided as a transgene to a mammal, preferably a mouse.
Also provided is a DNA-based assay to detect the presence of the ABri or ADan mutation in a subject suspected of harboring the mutation. The assay detects either a XbaI restriction site added as a result of the mutation, or the presence of additional DNA fragments compared to wild-type in a PCR amplified preparation of DNA.
Among the most preferred embodiments is a transgenic mammal, preferably a rodent, more preferably a mouse, having a diploid genome comprising a transgene encoding a heterologous amyloid precursor polypeptide having the British (ABriPP) or Danish (ADanPP) mutation, wherein the transgene is expressed to produce a human precursor polypeptide having the indicated mutation, and wherein the polypeptide is processed to the 34 amino acid amyloid peptide ABri or ADan, respectively in a sufficient amount to be detectable in the brain of the transgenic rodent.
Expression of the DNA constructs of the invention as a transgene in the mammal, preferably mouse, results in brain amyloid deposits, neurofibrillary tangles, non-neuritic plaque formation degeneration of neurons in the hippocampus and/or cerebellum; the mice exhibit cognitive impairment. The transgene is integrated in somatic and germ cells. The above transgene may be nonhomologously integrated.
The above construct is transcribed in brain cells of the mouse to form mRNA which is translated into detectable levels of ABriPP or ADanPP protein.
Also included are progeny of the above transgenic mice, wherein the genome of the progeny animal comprises the ABriPP or ADanPP transgene comprising operatively linked regulatory sequences as described below, wherein neural expression of the transgene produces impaired performance of the progeny mice in memory and learning tests and induces abnormal neuropathology as described below, in the progeny""s brain, wherein the impaired performance and the abnormal neuropathology are in comparison with control mice. The transgenic mouse may be one wherein its nontransgenic ancestor is from a strain having greater longevity as compared with other strains of mice.
As a result of the expression of the transgene, the transgenic mouse preferably suffers from progressive neurological disease characterized as an age-dependent decline in performance in memory and learning tests, non-neuritic amyloid plaques that are detectable by Congo red staining in the mouse""s brain, wherein the decline in performance and amyloid plaques are in comparison with control mice.
The transgenic animals produced in accordance with the present invention are intended to provide an experimental medium for elucidating aspects of the molecular pathogenesis of FBD, FDD or other neurodegenerative diseases that include dementia, and to serve as tools for screening drugs that may have potential application as therapeutic agents to prevent or limit amyloid accumulation, development of neurofibrillary tangles, neuronal degeneration, and therefore reduce or prevent the behavioral and physical symptoms consequent to such pathophysiology.
The invention includes cells from the above transgenic mouse or progeny, which cells comprise the mutant transgene DNA and in which expression of the DNA construct results in the production of detectable levels of the precursor proteins or amyloid peptides thereof.
This invention includes a method for enhancing neurodegeneration in the brain of a transgenic mouse, the method comprising:
(a) introducing the above DNA expression construct into a mouse embryo thereby producing a transgenic mouse embryo whose genome comprises the DNA sequence;
(b) developing the transgenic mouse embryo into a mouse, wherein expression of the DNA sequence results in an earlier onset of neurodegeneration in the brain of the transgenic mouse as compared to a control transgenic mouse that expresses (i) the wild-type human BriPP or (ii) endogenously expresses the murine homologue of BriPP.
The present invention is further directed to a method for identifying an agent that inhibits neurodegeneration in FBD, FDD or in a neurodegenerative disease having the symptoms of amyloid deposition, non-neuritic plaque formation, neurofibrillary tangles, the method comprising;
(a) providing a transgenic mouse as described above,
(b) administering to the mouse a candidate agent; and
(c) assaying or observing a decrease in neurodegeneration, or any of the indicated neuropathological signs, in the brain of this mouse as compared to neurodegeneration or neuropathological signs in the brain of a mouse of step (a) that is not administered the compound.
The decreased neuropathologic findings as a result of administering an agent that is positive in the transgenic model includes one or more of: reduction in number of ABri or ADan amyloid deposits; a reduction of hypertrophic gliosis in cortico-limbic structures; a reduction of diminished 2-deoxyglucose uptake or utilization in cortico-limbic structures of the brain.